1. Field of the Invention
The present invention is directed to a novel membrane protease isolated from malignant human melanoma cells such as the LOX line and RPMI7951 line. The invention is further directed to diagnosis of cellular transformation involving the use of this protease.
2. Background of the Invention
The invasion of tumor cells through surrounding connective tissue is an important stage of metastasis requiring cell surface protease activity (Jones, P. A., and DeClerck, Y. A. (1980), Cancer Res. 40, 3222-3227; Chen, W.-T., Olden, K., Bernard, B. A. and Chu, F.-F. (1984), J. Cell Biol. 98, 1546-1555). Tumor cell invasiveness has been linked with an increased production of extracellular matrix (ECM)-degrading enzymes, including plasminogen activators (for a review see Dano, K., Behrendt, N., Lund, L. R., Ronne, E., Pollanen, J., Salonen, E. M., Stephens, R. W., Tapiovaara, H., and Vaheri, A. (1989), Cancer Metastasis V. Schirrmacher R. Schwartz-Albiez (Ed.s) Springer Verlag Berlin, pp. 98-107), lysosomal cysteine proteases (Poole, A. R., Tiltman, K. J., Recklies, A. D., and Stoker, T. A. (1980), Nature 273, 545-547; Sloane, B. F., Rozhin, J., Johnson, K., Taylor, H., Crissman, J. D., and Honn, K. V. (1986), Proc. Natl. Acad. Sci. U.S.A., 83, 2483-2487), collagenases (Liotta, L. A., Abe, S., Robey, P. G., and Martin, G. R. (1979), Proc. Natl. Acad. Sci., U.S.A., 76, 2268-2272), and glycosidases (Nakajima, M., Irimura, T., DiFerrante, N., and Nicolson, G. L. (1984), J. Biol. Chem. 259, 2283-2290). Very little attention, however, has been paid to membrane proteases.
Plasma membrane-associated proteases have been identified from biochemically defined "membrane" fractions, which were derived from mixed cell types in tissues (Almenoff, J., and Orlowski, M. (1983), Biochemistry 22, 590-599; Malfroy, B., Schofield, P., Kuang, W. J., Seeburg, P. H., Mason, A. J., and Henzel, W. J. (1987), Biochem. Biophys. Res. Commun. 144, 59-66; Tanaka, K., Nakamura, T., and Ichihara, A. (1986), J. Biol. Chem. 261, 2610-2615). Studies from some tumors have shown that solid tumors contain heterogeneous cell types, and invading tumor cells represent less than 1/10,000 of the population (Poste, G., Doll, J., Hart, H., and Fidler, I. J. (1980), Cancer Res. 40, 1636-1644). Thus, attempts to identify these proteases from in situ solid tumors have not been successful.
Isolation of membrane proteases from tumor cells in culture that contain a homogeneous population, has shown some important results. Cell fractionation has localized cathepsin B-like activity to the plasma membrane of tumor cells (Sloane, B. F., Rozhin, J., Johnson, K., Taylor, H., Crissman, J. D., and Honn, K. V. (1986), Proc. Natl. Acad. Sci. U.S.A., 83, 2483-2487). A chymotrypsin-like protease activity was also identified from tumor cell membranes (Yavelow, J., Caggana, M., and Beck, K. A. (1987), Cancer Res. 47, 1598-1601; Zucker, S., Wieman, J. M., Lysik, R. M., Wilkie, D., Ramamurthy, N. S., Golub, L. M. and Lane, B. (1987), Cancer Res. 47, 1608-1614). In addition, the 100 kD common acute lymphoblastic leukemia antigen (CALLA) has been identified as neutral endopeptidase 24.11, an integral, zinc metalloendoproteinase (Shipp, M. A., Vijayaraghavan, J., Schmidt, E. V., Masteller, E. L., D'Adamio, L., Hersh, L. B., and Reinherz, E. L. (1989), Proc. Natl. Acad. Sci. U.S.A., 86, 297-301), which may be identical to the neutral metalloendoprotease purified from the detergent-extracted membrane fraction of rabbit kidney tissue (Almenoff, J., and Orlowski, M. (1983), Biochemistry 22, 590-599).
Previously, plasminogen activator was localized on the cell surface of Rous sarcoma virus (RSV)-transformed cells (Quigley, J. P. (1976), J. Cell Biol. 71, 472-486) and was shown to be involved in tumor metastasis (Ossowski, L., and Reich, E. (1983), Cell 35, 611-619). L. B. Chen and Buchanan (Chen, L. B., and Buchanan, J. M. (1975), Proc. Nat. Acad. Sci. U.S.A., 72, 1132-1136), however, showed that plasminogen-independent fibrinolysis of cell surface proteins involved unidentified proteases produced by transformed cells. It has also been shown that RSV-transformed cells express invasiveness by locally degrading fibronectin crosslinked gelatin films at rosette contact sites or invadopodia (Chen, W.-T., Olden K. Bernard, B. A. and Chu, F.-F. (1984), J. Cell Biol. 98, 1546-1555; Chen, W.-T., Chen, J. M., Parsons, S. J., and Parsons, J. T. (1985), Nature (Lond.) 316, 156-158; Chen, W.-T. (1989), J. Exptl. Zool. 251, 167-185). The transformed cells express 120 kD and 150 kD proteases in association with the membrane, which degrade fibronectin (Chen, J.-M. and Chen, W.-T. (1987), Cell 48, 193-203). These proteases are present in extremely low quantity and the possibility that these proteases are associated with viruses in the RSV transformation model is difficult to rule out.
Crosslinked gelatin films comprising fluorescent and radiolabeled proteins covalently coupled to the surface of the substratum have been developed (Chen, W.-T., Olden, K., Bernard, B. A. and Chu, F.-F. (1984), J. Cell Biol. 98, 1546-1555). This technique allows identification of tissue culture cells which express activated ECM-degrading proteases at sites of contact between cells and their substrata.